OsCc1 promoter and methods of transforming monocot plants using the same

ABSTRACT

The present invention relates to a process for varying a trait of monocot plants, wherein the process comprises the step of transforming a monocot plant with a recombinant plasmid containing an OsCc1 promoter and a desired nucleic acid.

FIELD OF THE INVENTION

The present invention relates to a process for varying a trait ofmonocot plants, which comprises steps of transforming a monocot plantwith a recombinant plasmid containing OsCc1 promoter for transformationof monocot plants and a desired foreign gene and of expressing saidforeign gene.

BACKGROUND THE INVENTION

In producing farming plants having novel characteristics, which candevelop an agricultural field, expression of a foreign gene (i.e.,transgene) to be introduced into a plant body is greatly influenced bytranscriptional, post-transcriptional, translational andpost-translational elements. Among said elements, particularly, apromoter belonging to the transcriptional elements is the most importantelement, which not only can directly influence a transcription of atransgene to ultimately change the expression level, but also can changethe steps of expressing a transgene, or the tissue- orcell-specificities. To date, although numerous promoters have beenisolated from various plants for expression of a trangene, only a fewpromoters among them are currently practically used for transformationof a plant body. CaMV (cauliflower mosaic virus) 35S promoter and itsderivatives, which have been most widely used in this field at present,induce an extensive expression of genes in the whole tissues of a plantbody and, exhibit a high activity especially in vascular tissues andmost cells of roots and leaves. However, the CaMV 35S promoter exhibitslower activity in monocot plants such as rice plant, etc., than in dicotplants, and even does not exhibit any activity in certain cells such aspollen, etc. Numerous promoters other than CaMV 35S promoter, which haveoriginated from dicot plants, have also been used for transformation ofmonocot plants, but exhibit lower activity than do promoters originatingfrom monocot plants. Further, rbcS (ribulose bisphosphate carboxylase/oxygenase small subunit) promoter of rice plant, Act1 (actin1) promoterof rice plant, and Ubi1 promoter of maize plant have been investigatedas a promoter useful for transformation of monocot plants. Among them,Act1 and Ubi1 promoters exhibit relatively a high activity in monocotplants as compared to CaMV 35S promoter, and therefore, have beengenerally used for transformation of monocot plants. However, Ubi1promoter exhibits activity in numerous types of cells but does not coverthe whole tissues of a plant body. Moreover, although Ubi1 promoterexhibits a strong activity especially in young roots, its activity isgreatly reduced as the roots grow. In addition, Act1 promoter exhibitsactivity mainly in the elongating tissues and reproductive tissues.Thus, said promoters are not effective for expression of a ubiquitousgene in monocot plants.

Therefore, there exists a continuous necessity for developing a promotershowing a strong, stable and ubiquitous activity in transforming monocotplants.

SUMMARY OF THE INVENTION

According to the present invention, a rice plant was transformed with arecombinant plasmid containing a promoter for rice cytochrome c gene(OsCc1) and sgfp gene encoding a transformed green fluorescent protein,and the expressed fluorescent protein was then analyzed; as a result, itwas identified that OsCc1 promoter can strongly, stably and ubiquitouslyinduce the expression of a foreign gene in all the tissues of the riceplant. Therefore, OsCc1 promoter can be very effectively used fortransformation of monocot plants.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 a, 1 b and 1 c are photographs showing the results of Nothernblot analysis using total RNAs extracted from various tissues of anuntreated rice plant, various tissues of a rice plant treated withBt₂cAMP, and leaves of young seedlings treated with light, respectively;

FIG. 2 shows gene maps of plasmids pSB-CG, pSB-RG and pSBG700;

FIG. 3 is a photograph showing the result of Western blot analysis forproteins extracted from various tissues of a transformed rice plant;

FIG. 4 a is a photograph showing sGFP fluorescent images of youngseedlings transformed with OsCc1-sgfp (OsCc1), Act1-sgfp (Act1) orrbcS-sgfp (rbcS);

FIG. 4 b shows photographs of sGFP fluorescent images of various tissuesof rice plants transformed with OsCc1-sgfp (OsCc1), Act1-sgfp (Act1) orrbcS-sgfp (rbcS); and

FIG. 4 c shows graphs of relative sGFP fluorescent intensity of varioustissues of rice plants transformed with OsCc1-sgfp (OsCc1), Act1-sgfp(Act1) or rbcS-sgfp (rbcS).

DETAILED DESCRIPTION OF THE INVENTION

Technical Field to which the Invention Belongs and the Prior Art in thisField

The present invention relates to a promoter for transformation ofmonocot plants. More specifically, the present invention relates to aprocess for varying a trait of monocot plants which comprises steps oftransforming a monocot plant with a recombinant plasmid containing OsCc1promoter for transformation of monocot plants and a desired foreign geneand of expressing said foreign gene.

Technical Subject to be Accomplished by the Invention

In response to said necessity in this technical field, the presentinventors have taken a strong effort to develop a promoter effective fortransformation of monocot plants. As a result, we have identified thatwhen a rice plant is transformed with a recombinant plasmid containing apromoter for rice cytochrome c gene (OsCc1) and sgfp gene coding for atransformed green fluorescent protein and then the expressed fluorescentprotein is analyzed, the fluorescent protein is strongly, stably andubiquitously expressed in the whole tissues of the rice plant, andaccordingly, we came to complete the present invention.

Therefore, the main purpose of the present invention is to provide OsCc1promoter for transformation of monocot plants.

Another purpose of the present invention is to provide a process forvarying a trait of monocot plants, which comprises transforming amonocot plant with a recombinant plasmid containing OsCc1 promoter.

[Constitution of the Invention]

In order to develop a promoter effective for transformation of monocotplants, the present inventors have first paid attention to cytochrome c.Cytochrome c is a small protein located in mitochondria of eukaryotes,and functions as a catalyst for transfer of electrons between therespiratory complexes III and IV. Up to now, cytochrome c has beenextensively used for molecular evolutionary studies because its aminoacid sequences and base sequences are highly preserved even among theorganisms distantly related such as yeasts, mammals and plants. Further,it has also been used as a molecule useful for a protein targeting intoorganelles because of its unique subcellular location. Recently,cytochrome c has attracted a keen attention because its release intocytosol is an indicator of apoptosis in human cells. However, little isknown in plants about cytochrome c except that Arabidopsis and ricecytochrome c genes (OsCc1) were cloned and sequenced (see, Kemmerer etal., Mol. Biol. Evol., 8:212-226, 1991).

Thus, the present inventors have expected that since cytochrome c isinvolved in the electron transfer to play a role of supplying energy tocells, cytochrome c gene would be expressed at a high level in cells,and continuously and stably expressed in the whole tissues ofindividuals regardless of the development stage. Accordingly theinventors intended to develop OsCc1 promoter in the form of a promoterwhich can induce expression of said gene at a high level in the wholetissues of a plant body. Particularly, since in most cases promotersutilized for transformation of plants cannot be commonly used in dicotplants and monocot plants, they should be independently developed inboth plants. However, at present the promoters which can be used formonocot plants are very limited, such as, Ubi1 and Act1 promoters, etc.,and therefore, we have put the main emphasis on the development of OsCc1promoter in the form of a promoter effective for transformation ofmonocot plants.

Hereinafter, the present invention will be more specifically explained.

The present invention provides a process for varying a trait of monocotplants which comprises steps of transforming a monocotyledon with arecombinant plasmid containing OsCc1 promoter for transformation ofmonocot plants and a desired foreign gene and of expressing said foreigngene. In this process, the foreign gene may be all the genes which areintended to be expressed at a high level in the whole tissues of a plantbody, including selective marker genes comprising hygromycin-resistantgene Hyg^(R) and herbicide-resistant gene bar or genes resistant tobiological or non-biological stresses. The monocot plants include rice,barley, wheat or maize plants; and the transformation can beaccomplished by particle bombardment method or Agrobacterium-mediatedmethod.

The present inventors first conducted Nothern blot analysis for a totalRNA extracted from respective tissues of a rice plant to analyzeexpression pattern of OsCc1 gene; constructed a recombinant plasmidcontaining OsCc1 promoter and sgfp gene coding for transformed greenfluorescent protein; transformed a rice plant with said recombinantplasmid via Agrobacterium-mediated method; then, conducted Southern blotanalysis and Western blot analysis using the gene and the proteinextracted from the transformed rice plant, respectively; and analyzedthe fluorescent image of the rice plant to determine the activity ofOsCc1 promoter. It has been known that since OsCc1 promoter of animalscontains CRE-binding site (cAMP response element binding site), itsactivity is greatly influenced by the concentration of cAMP; rbcSpromoter having a high activity specifically in chloroplast is greatlyinfluenced by light due to its unique properties; and OsCc1 promoter ofa rice plant contains the base sequence similar to that of CRE-bindingsite. Therefore, said Nothern blot analysis was conducted to determinewhether OsCc1 promoter which leads the expression of OsCc1 gene isinfluenced by cAMP or light. The activity of OsCc1 promoter was comparedwith the activities of Act1, rbcS and Ubi1 promoters, which arecurrently practically used for transformation of monocot plants.

The present invention will be more specifically illustrated through thefollowing examples. A person having an ordinary knowledge in thistechnical field can clearly understand that these examples are intendedonly to specifically explain the present invention and the scope of thepresent invention is not limited to these examples in any manner.

EXAMPLE 1 Expression Pattern of OsCc1 gene

In order to investigate the expression pattern of OsCc1 gene, a totalRNA was extracted from the respective tissues and cells of a rice plantby guanidinium/LiCl method and then subjected to Nothern blot analysis.First, embryogenic calli of a rice plant were obtained from embryo ofmature Oryza sativa L. (cv Nakdong) and then maintained in MS solidmedium, pH 5.8, containing 1% (w/v) agarose, 30 g/L sucrose and 2.5 mg/L2,4-D. Said embryogenic calli were incubated in AA liquid mediumcontaining 30 g/L sucrose, 2.0 mg/L 2,4-D and 0.2 mg/L kinetin using ashaking incubator at 120 rpm and in the dark at 26 to obtain suspensioncultured cells. Rice plants, suspension cultured cells and calli, withor without treatment by 1 mM dibutyl cAMP (Bt₂cAMP, Sigma, USA) or bylight, were frozen in liquid nitrogen. About 0.1 g of leaves, roots,calli and young seedlings were homogenized in 1 ml of extraction buffersolution containing 4 M guanidinium isothiocyanate, 25 mM sodium citrate(pH 7.0), 0.5% (w/v) sarcosyl and 0.1 M β-mercaptoethanol, then added0.1 ml of 2 M sodium acetate (pH 4.0), 1 ml of water-saturated phenol(pH 4.5) and 0.2 ml of chloroform:isoamyl alcohol (1:1, v/v), well mixedtogether for 30 seconds and centrifuged at room temperature for 10minutes at 5,000 g. The equal volume of isopropanol was added to thesupernatant obtained from centrifuge, and then the mixture was allowedto stand for one hour at −20, thereafter centrifuged at 4 for 10 minutesat 10,000 g, washed with 70% (v/v) ethanol and dried. The resulting RNApellet was dissolved in 40 ml of DEPC (diethyl pyrocarbonate)-treatedwater and the concentration of RNA was calculated from A₂₆₀ value. Then,Nothern blot analysis was conducted using 20 μg of total RNA accordingto the method of Sambrook, et al., (see, Sambrook J. et al., MolecularCloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor LaboratoryPress, 1989). RNA transferred to the membrane was hybridized to[³²P]-probe present in a random primer labeling kit (Takara, Japan),washed and then analyzed by a phospho-image analyzer (FLA 3000, Fuji,Japan).

FIGS. 1 a, 1 b and 1 c are photographs showing the results of Nothernblot analysis for total RNAs extracted from various tissues of untreatedrice plant, from various tissues of rice plant treated with Bt₂cAMP, andfrom leaves of young seedlings treated with light, respectively. InFIGS. 1 a and 1 b, the symbols SC, L, R and C represent suspensioncultured cells, leaves, roots and callus, respectively; the numericalvalues in FIGS. 1 b and 1 c represent the times during which the testsamples were treated with Bt₂cAMP or light; and the symbol L in FIG. 1 crepresents mature leaves. As can be seen from FIGS. 1 a, 1 b and 1 c,OsCc1 was expressed at a high level in suspension cultured cells, rootsand callus, but at a relatively low level in leaves, and further theexpression of OsCc1 was not influenced by cAMP or light. Therefore, itcan be said that OsCc1 is independent on cAMP or light and is expressedin non-photosynthetic tissues at a higher level than in photosynthetictissues. This result is consistent with the fact that chloroplastfunctions to supply major energy in photosynthetic tissues whereasmitochondria does in non-photosynthetic tissues.

EXAMPLE 2 Construction of Plasmid

Plasmids containing promoter of OsCc1, Act1 or Ubi1 and sgfp gene codingfor transformed green fluorescent protein were constructed. 1.8 kb ofOsCc1 promoter (SEQ. ID. No. 1) was PCR amplified from pOsCc1 (see,Kemmerer EC. et al., Mol. Biol. Evol., 8:212-226, 1991) using thefollowing two primers a and b containing XhoI or NcoI restriction site(indicated by underline):

5′-AACTGGAGGAATTCGGATCTTCGAAGGTAGGC-3′; primer a (SEQ. ID. No. 2) and5′-AACCATGGCCGCCGCCGCCGCGAGAACG-3′; primer b (SEQ. ID. No. 3).

The amplified DNA was digested with XhoI and NcoI and then ligated topBluescript KSII (see, Chiu W-L, et al., Curr. Biol., 6:325-330, 1996)containing sgfp gene digested with the same restriction enzymes toconstruct plasmid pKSCG. Thereafter, the 2.5-kb DNA fragment containingthe OsCc1-sgfp was obtained by digestion of pKSCG with XhoI and NotI,and ligated to pSB 105 (see, Jang, I-C. et al., Mol. Breeding,5:453-461, 1999) digested with the same restriction enzymes to producethe plasmid pSB-CG (OsCc1-sgfp). pSB 105 contains potato proteaseinhibitor II gene terminator, ³⁵S promoter, bar gene (phosphinothricinacetyltransferase gene) and nopaline synthase gene terminator betweenthe right-border sequence and the left-border sequence of pSB11.Phosphinothricin acetyltransferase encoded by bar gene plays a role ofdetoxifying phosphinothricin-based herbicides and therefore, can serveas a selective marker. Then, the DNA fragment was obtained by digestionof pSK-RG (see, Chiu W-L, et al., Curr. Biol., 6:325-330, 1996),containing rbcS promoter linked to sgfp gene, with BamHI and NotI, andligated to pSB105 digested with the same restriction enzymes to producethe plasmid pSB-RG (rbcS-sgfp). Thereafter, the rbcS promoter in theresulting plasmid pSB-RG was replaced with Act1 promoter of rice (see,McElroy D. et al., Mol. Gen. Genet., 231:150-160, 1991) to produceplasmid pSBG700 (Act1-sgfp).

FIG. 2 is the drawing showing the gene maps of plasmids pSB-CG, pSB-RGand pSBG700. In FIG. 2, BR and BL represent the right border and theleft border, respectively; and 3′PinII and 3′ Nos represent terminatorsof potato protease inhibitor II gene and of nopaline synthase gene,respectively. Plasmids pSB-CG, pSB-RG and pSBG700 constructed as abovewere respectively introduced into Agrobacterium tumefaciens LBA4404 bytriparental mating.

EXAMPLE 3 Stability of Transgene in a Rice Plant Produced byAgrobacterium—Mediated Transformation

To about 200 non-trashed grains (Oryza sativa L. cv Nakdong) was added70% (v/v) ethanol and slightly mixed for one minute to sterilize thegrains. Next, ethanol was discarded and the residual grains wereslightly mixed with 100 ml of 20% (v/v) CLOROX for one hour to furthersterilize the grains, which were then washed several times withsterilized water. In order to transform a rice plant, callus induction,co-cultivation with Agrobacterium containing the plasmid as prepared inthe above example and selection of transformed callus were conductedaccording to the method described by Jang, et al. (see, Jang, I-C. etal., Mol. Breeding, 5:453-461, 1999). The rice plant transformed byAgrobacterium-mediated method was grown in a greenhouse to select onlythe rice plant having a resistance to herbicide. By Southern blotanalysis of the genome of the transgenic rice plant as transformed andselected as above, it was identified that transduced transgene wasintegrated into chromosome of the rice plant and had 1 to 3 copies. Inview of the fact that a rice plant was identified as containing a singlecopy of OsCcl in its genome by genomic DNA hybridization, we couldeasily expect that the transgene would be stably located in a plant bodyand expressed at a high level.

EXAMPLE 4 Activity of Promoters in the Respective Tissues of aTransformed Rice Plant

By analysis of fluorescent proteins expressed in the respective tissuesof a rice plant transformed with plasmids pSB-CG, pSB-RG and pSBG700constructed as above, the activities of the respective promoters wereanalyzed and compared with each other.

EXAMPLE 4-1 Western Blot Analysis of Fluorescent Proteins

Proteins were extracted from the respective tissues of a transformedrice plant and then subjected to Western blot analysis using sGFPantibody. About 0.1 g of callus, leaf and root tissues of a rice plantwere ground in liquid nitrogen, homogenized in buffer solutioncontaining 20 mM Tris-HCl (pH 8.0), 10 mM EDTA, 30 mM NaCl and 2 mMphenylmethanesulfonyl fluoride at 4 for one hour, and then centrifugedat 4 for 5 minutes. The concentration of protein contained in thesupernatant was determined using Bradford solution (Bio-Rad, USA). Crudeprotein was electrophoresed on 15% SDS-polyacrylamide gel,electroblotted to polyvinylidene difluoride (PVDF) by means of Semidryapparatus (Bio-Rad, USA), and then subjected to immunoblot analysisusing sGFP polyclonal antibody (Clontech, USA) and the secondaryantibody conjugated with alkaline phosphatase. FIG. 3 is the photographshowing the result of Nothern blot analysis for proteins extracted fromthe respective tissues of a transformed rice plant. Proteins wereextracted from 2, 3 or 3 plants selected from rice plants transformedwith OsCc1-sgfp, Act1-sgfp or rbcS-sgfp, respectively, and thenanalyzed. In FIG. 3, the symbols L, R and C, PC and NC representproteins extracted from leaves, roots, callus, purified sGFP, and leavesof untransformed rice plant, respectively, and the respective numericalvalues denote the number of selected individuals. From FIG. 3, it couldbe seen that sGFP is expressed in all the tissues of a rice planttransformed with OsCc1-sgfp at a high level, which is similar to thosein rice plants transformed with Act1-sgfp or rbcS-sgfp.

EXAMPLE 4-2 Fluorescent Analysis of sGFP in a Transformed Rice Plant

In order to examine the activity of OsCc1 promoter at the level of thewhole plant, the transformed seeds and the untransformed seeds weregrown to analyze the fluorescence of sGFP in a rice plant. Ahigh-resolution CCD color video camera (Model CoolSNAP, Roper ScientificInc., USA) utilizing a digital video imaging system was used to collectsGFP fluorescent images of young seedlings, transformed with OsCc1-sgfp(OscC1), Act1-sgfp (Act1) or rbcS-sgfp (rbcS), in a perpendicularposition. The result obtained is shown in FIG. 4 a. In addition, sGFPfluorescent images in leaves, root apexes, elongating regions of rootsand hairy roots of the respective transformed rice plants were observedwith confocal laser scanning microscopy (Carl Zeiss LSM510, Carl Zeiss,Germany). The observation result is shown in FIG. 4 b. The intensity ofsGFP fluorescence observed in FIG. 4 b was calculated by means of asoftware equipped in LSM510 version 2.8 and the relative fluorescentintensity thereof, which was calculated on the basis of the value of thefluorescent intensity of MC developed by Act1 activity to be fixed as 1,is shown in FIG. 4 c. In FIG. 4 a the symbol NT represents untransformedyoung seedlings; in FIG. 4 b the symbols GC, RA, RC, RHP, LC, SC, PR andHR represent guard cells, root apexes, root caps, hair protuberances ofroot, long cells, short cells, primary roots and hairy roots,respectively (where the scale bar represented by the white line has alength of 30 μm); and in FIG. 4 c the symbols MC, RA and RE representmesophyll cells, root apexes and elongating regions of roots,respectively.

From FIG. 4 a, it could be seen that untransformed young seedlings(NT)do not show any fluorescence whereas transformed young seedlings (OsCc1,Act1 and rbcS) exhibit light green fluorescence and particularly, OsCc1exhibits fluorescence in whole tissues of the plants. In addition, fromFIG. 4 b, in leaf cells, it could be seen that plants transformed withOsCc1-sgfp exhibit sGFP fluorescence at a higher level than plantstransformed with Act1-sgfp but at a lower level than plants transformedwith rbcS-sgfp. In root cells, it could be seen that plants transformedwith OsCc1-sgfp or Act1-sgfp exhibit sGFP fluorescence at a higher levelthan plants transformed with rbcS-sgfp, and particularly, plantstransformed with OsCc1-sgfp exhibit brighter fluorescence in root apex,root cap and hair protuberance of roots. Therefore, it can be regardedthat OsCc1 promoter exhibits the activity in the whole tissues of aplant body and particularly, displays a higher activity in rapidlydividing cells and cells having a high metabolic activity. Such resultcould also be identified from FIG. 4c. The following Table 1 shows theactivities of OsCc1 promoter and the promoters which are widely used fortransformation of rice plants. In Table 1, 35S and 35Si denoteintron-free CaMV 35S promoter and CaMV 35S promoter conjugated withintron of maize Adh1 promoter, respectively; Adh1, Ubi1 and Act1 denotepromoters conjugated with their intrinsic introns, respectively; andOsCc1 and rbcS denote intron-free rice promoters. The values shown inthe table is a relative value calculated on the basis of the value ofthe activity of CaMV 35S or Act1 to be fixed as 1. From Table 1, itcould be seen that the activity of OsCc1 promoter in leaves and roots ishigher than that of Act1 promoter by 5 and 2 times, respectively.

TABLE 1 Activities of OsCc1 promoter and the promoters which are widelyused for transformation of rice plants 35 35S Adh Ubi Act rbc OsCcAnalysis S i 1 1 1 1 1 Remarks Temporary analysis^(a) 1 4.2 4 ND 22 NDND M Temporary analysis^(b) ND 1 1.5 10 1.5 ND ND C Leaves of transgenicplant ND ND ND ND 1 5 ND J Leaves of transgenic plant ND ND ND ND 1 12 5I Roots of transgenic plant ND ND ND ND 1 0.5 2 I Notes) ^(a)protoplastwhich originated from suspension cultured cells ^(b)protoplast whichoriginated from callus cells M: McElroy D. et al., Mol. Gen. Genet.,231: 150-160, 1991 C: Cornejo M-J. et al., Plant Mol. Biol., 23:567-581, 1993 J: Jang I-C, et al., Mol. Breeding, 5: 453-461, 1999 I:the present invention ND: the value is not determined.Thus, while the invention has been particularly shown and described withrespect to preferred examples thereof, it will be understood by thoseskilled in the art that changes in forms and details may be made thereinwithout departing from the scope and spirit of the invention.

1. An isolated OsCcl promoter of SEQ ID NO:1 for transformation ofmonocot plants.
 2. A process for varying a trait of a monocot plant,wherein the process comprises transforming a monocot plant with arecombinant plasmid containing an OsCcl promoter operably linked to adesired isolated nucleic acid sequence, whereby a trait of a monocotplant is varied.
 3. The process for varying trait of a monocot plantaccording to claim 2, wherein the isolated nucleic acid sequence is aselective marker gene comprising Hyg^(R) and bar, or provides resistanceto biological or non-biological stresses.
 4. The process for varying atrait of a monocot plant according to claim 2, wherein the monocot plantis a rice, barley, wheat or maize plant.
 5. The process for varying atrait of a monocot plant according to claim 2, wherein transforming isaccomplished by a particle bombardment or is Agrobacterium-mediated.